In prokaryotes, cAMP is known to regulate the transcription of specific genes by a well-defined mechanism. cAMP binds to the prokaryotic cAMP receptor protein (CRP), and this complex interacts with specific DNA sequences at the 5'ends of targeted genes, modulating the ability of RNA polymerase to bind to the pro- moters. cAMP has also been shown to alter the transcription of specific eukaryotic genes. Recent evidence suggests a direct action of cAMP on mammalian genes in which the regulatory subunit (R) of cAMP-dependent protein kinase might interact directly with target genes. Nagamine and Reich proposed that the R subunit of cAMP-dependent protein kinase could be envisaged as a cAMP- and DNA-binding molecule which regulates gene transcription, the vertebrate counterpart of bacterial CRP. However, this hypothesis lacked evidence for a direct interaction between DNA and cAMP- dependent protein kinase or the R subunit. In this study, we examined the role of cAMP receptor protein, R, in gene transcription. Since cAMP-dependent protein kinase is exclusively present in the cytoplasm, any nuclear function of the protein kinase must be accompanied by its translocation to the nucleus. It was found that the ras gene suppression, growth inhibition, and phenotypic reversion of Ha-MuSV-transformed NIH/3T3 cells by site- selective cAMP analogs correlated with the nuclear translocation of the RII cAMP receptor protein, the regulatory subunit of protein kinase type II. Within 30 min after treatment of the transformed cells with the analogs, immunofluorescence against the RII protein markedly increased in the cell nucleus. Thus, the nuclear translocation of the RII cAMP receptor protein is an early event in the reverse transformation induced by the cAMP analogs. The goal of this study is to provide direct evidence of interaction between the cAMP consensus sequences and cAMP receptor protein (RII) and/or a phosphoprotein substrate of protein kinase.